Evidence has emerged for a direct role of glucose, independent of insulin, in the regulation of cellular glucose transport and glucose utilization in vivo. In this study, we have investigated potential cellular and molecular mechanisms for this regulatory effect of glucose by determining how normalization of glycemia without insulin therapy in diabetic rats influences 3-O-methylglucose transport and the expression and translocation of two genetically distinct species of glucose transporters (GTs) in adipose cells. The data show that normalization of blood glucose in diabetic rats with phlorizin, which impairs renal tubular glucose reabsorption and thus enhances glucose excretion, restores insulin- stimulated glucose transport in adipose cells and insulin-mediated glucose disposal in vivo and thus that ambient glucose independent of ambient insulin can regulate the glucose transport response to insulin in isolated adipose cells and changes in responsiveness parallel alterations in in vivo glucose uptake. Since this effect can occur without alteration in the expression of the two species of glucose transporters present in adipose cells or in their translocation to the plasma membrane in response to insulin, it may result from changes in GT functional activity.